The T24 cell line has been established from a highly malignant grade III human urinary bladder carcinoma [20]. This cell line can be easily grown in vitro and has been extensively used to evaluate the therapeutic effects of several anticancer drugs. Here, we describe the MK-2206 mw preliminary results of

the study of the therapeutic effect of sirolimus against human T24 bladder cancer cell line in vitro using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for assessing cell proliferation and Trypan blue for assessing cell viability. Materials and methods Cell culture Cell line T24 was provided by a German collection of microorganisms and A-1210477 manufacturer cell cultures (DSMZ, Düsseldorf, Germany). Cells were grown as a monolayer in complete RPMI (RPMI-1640 medium supplemented with 10% fetal calf serum, 100 U/mL penicillin and l00 μg/mL Captisol streptomycin), in a humidified atmosphere with 7% CO2-93% air at 37°C. Under these conditions, the plating efficiency was 70–90% and

the doubling time was 9–10 h. Single cell suspensions were obtained by trypsinization of monolayer cultures. Drugs Sirolimus was purchased from Wyeth (Rapamune). Cell proliferation The anti-proliferative capacity of the treatments was assessed by the MTT [21]. This is based on the reduction of MTT by mitochondrial dehydrogenase of intact cells to a purple formazan product. Using a Neubauer counting chamber cells were counted and 2 × 104 cells were seeded in 1 ml of medium in a 96-well culture plates and allowed to attach for 24 hours. Oxalosuccinic acid Cells were treated with sirolimus (5 ng/mL, 10 ng/mL, 40 ng/mL, 60 ng/mL, 100 ng/mL,

150 ng/mL, 200 ng/mL, and 250 ng/mL) for 72 h, these doses were based on results published by other researchers [22, 23]. Each of the concentrations above was regarded as one treated group while there was no sirolimus in the control group. After incubation, cell proliferation was evaluated by MTT assay according to the manufacturer’s instructions. The MTT solution (20 μL, 5 mg/ml) was added to each well 3 h prior to the end of the 72 h chemical treatment exposure period. The media were removed at the end of the 72 h exposure period. The insoluble purple formazan crystals were dissolved in 100 μL DMSO/well and the absorbance was detected at 570 nm and 690 nm using a spectrophotometer (U 2000, Hitachi). The proliferation inhibitory rate percentage was calculated as follows: proliferation inhibitory rate (%)= 1-(A570-A690) of experimental wells/(A570-A690) of control wellsX100. Assays were performed in triplicate. Assay of cell viability The viability of T24 cell line was determined by Trypan blue exclusion analysis. 0.2 ml of the cells suspension treated with sirolimus at various concentrations were transferred to test tubes with 0.5 ml of 0.4% Trypan blue solution and 0.3 ml of HBSS and mixed thoroughly. Allow to stand for 5 to 15 minutes.